The epidermal growth factor receptor (EGFR) is a transmembrane protein expressed in epithelial surfaces. It plays an important physiological role in epithelial repair and regeneration. Epidermal growth factor (EGF) is a peptide secreted by salivary glands and other glands associated with epithelial surfaces that binds to a specific area in the extracellular domain of EGFR. Upon binding it generates a signal that is transmitted inside the cell. The first intracellular event as a result of EGF binding is a conformational change of the intracellular domain of EGFR that allows adenosine 5′-triphosphate (ATP) to enter the so-called tyrosine kinase (TK) domain, a pocket that contains a tyrosine residue, and donate a phosphate group to the tyrosine residue. The intracellular EGFR carrying a phosphorylated tyrosine becomes capable of associating with other intracellular proteins and originates a series of biochemical reactions that propagate downstream through a very complex network. The best known arms of this network are the mitogen-activated protein kinase (MAPK) pathway, which results in tumor cell division upon activation, and the AKT pathway, which results in enhanced cell survival upon activation. The results of EGFR activation are therefore increased cell proliferation and enhanced cellular tolerance to different insults. Many tumors overexpress EGFR compared to adjacent normal tissues or the epithelial surface from which they originate or have a mutated version of EGFR, intrinsically activated or with an enhanced susceptibility to activation. Such overexpression is thought to be one of the many mechanisms by which tumor cells gain a growth advantage, a key characteristic of the malignant phenotype. Consequently, blocking the EGFR signaling pathway is thought to be a rational strategy for the treatment of many human malignancies. There are basically two ways to inhibit upstream the EGFR signaling pathway: 1) preventing EGF and other natural peptide ligands from binding to the extracellular EGFR domain by the use of specific monoclonal antibodies, and 2) preventing ATP and other phosphate donors from entering the TK pocket of the intracellular EGFR domain by the use of small molecules that structurally fit very well into the pocket (i.e., EGFR TK inhibitors such as gefitinib and erlotinib).
Lung cancer is the leading cause of cancer-related death and non-small cell lung cancer (NSCLC) accounts for about 85% of the cases. In one unique subset of NSCLC patients, lung cancer cells harbor activating mutations in epidermal growth factor receptor (EGFR) and addict to aberrant EGFR signaling for cell survival. Among the activating mutations, L858R mutation and LREA deletion in EGFR account for over 90% of drug-sensitive mutations and show increased binding affinity toward tyrosine kinase inhibitors (TKIs) compared to wild-type EGFR. The administration of TKIs successfully induces the intrinsic apoptosis pathways in EGFR-mutant lung cancer cells; however, the dose-limiting side effect such as skin rash and diarrhea are unavoidably triggered by the concurrent inhibition of wild-type EGFR signaling in normal cells. Moreover, despite the success of tyrosine kinase inhibitors at the beginning of NSCLC treatment, the acquired secondary mutation at the gatekeeper residue 790 of EGFR (T790M), which is found in 50% of drug-resistant patients, weakens the interaction between TKIs and EGFR. Dose-limiting toxicity and T790M-derived drug resistance are the main issues in NSCLC treatment which still remain to be solved.
Ribozymes are naturally-occurring RNA molecules that contain catalytic sites, making them more potent agents than antisense oligonucleotides. However, wider use of ribozymes has been hampered by their susceptibility to chemical and enzymatic degradation and restricted target site specificity. A new generation of catalytic nucleic acids has been described containing DNA molecules with catalytic activity for specific RNA sequences. These DNA enzymes exhibit greater catalytic efficiency than hammerhead ribozymes, producing a rate enhancement of approximately 10 million-fold over the spontaneous rate of RNA cleavage, offer greater substrate specificity, are more resistant to chemical and enzymatic degradation, and are far cheaper to synthesize. With rational design, nucleic acid agents able to act on specific mRNAs to silence the expression of target genes at transcript- or allele-specific levels have been exploited by many labs around the world for decades. Among them, DNAzymes have been comprehensively studied to silence various genes with promising results for use as therapeutic agents. The basic structure of DNAzymes consists of a catalytic domain flanked by two substrate binding arms with their sequences complementary to targeted mRNA sequence.
DNAzyme has shown different reaction rate toward different nucleotide composition at mRNA cleavage site. Besides, unlike siRNAs which requires Dicer protein to form RNA-induced silencing complex (RISC) for mRNA cleavage, divalent metal ions such as Mg2+ or Ca2+, which are abundant in cell cytosol, are sufficient for catalyzing DNAzyme function. Combining these factors together, DNAzymes are cheap, stable, and easy manipulated nucleic acid agents with high efficient mRNA cleavage activity and low non-specific toxicity in cancer therapy.
Gary Beale et al. provide some ribozymes and DNAzymes in inhibiting EGFR expression in A431 cells (Journal of Drug Targeting, August 2003 Vol. 11 (7), pp. 449-456). However, the prior art reference indicates the efficacy of these ribozymes and DNAzymes are less effective in inhibition. Crispin R. Dass et al. published a review article that documents the rise of DNAzymes in the fight against cancer and serves as a forecast for this promising biotechnology in this context (Mol Cancer Ther 2008; 7(2):243-51). US 20120225870 discloses that an anti-ErbB or anti-MET therapeutic may be an enzymatic nucleic acid such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. However, no substantial enzymatic nucleic acid is provided in this reference.
There is still a need to develop DNAzymes which are capable to effectively silence the expression of EGFR or overcome TKI resistance accompanied with lower unwanted side effects.